In passages 1 through 3, five mice were inoculated with each C. jejuni strain; ten mice were inoculated with each strain in Caspase activity passage 4. As noted below (Materials and Methods), in this series of experiments, mice in the first passage were inadvertently
shifted from diets containing ~12% fat to ~6% fat just prior to C. jejuni infection for the first passage. This error was not discovered until after the mice had been infected. A previous experiment that allowed a direct comparison of C. jejuni 11168 infected C57BL/6 IL-10-/- mice on the ~12% fat diet and adapted to the ~6% fat diet for at least two weeks prior to infection did not reveal a statistically significant difference in survival, gross pathology or histopathology (data not shown). Therefore, all subsequent passages included a similar dietary shift prior to inoculation in order to maintain constant dietary conditions in the mice across mTOR inhibitor the four serial passages. During the first three passages of the serial passage experiment, fecal C. jejuni populations were monitored by plating on C. jejuni selective medium; population sizes were scored on a semi-quantitative scale with ranks from 0 to 4  (Figure 2). Briefly, colonization was scored as 0 if plates had no C. jejuni cfu, level 1 if plates had < 20 cfu, level 2 if plates had > 20 but < 200 cfu, level 3 if plates had > 200 cfu, and
level 4 if plates were covered with a lawn of C. jejuni. Two-way ANOVA was performed on the ranked colonization data from the first three passages with the Holm-Šidák test for post hoc comparisons. For all strains except D0835, ranked population sizes varied with the day of sampling (P = 0.006 for strain click here 11168, 0.004 for strain D2586, 0.028 for strain D2600,
and 0.009 for strain NW). In the four strains where significant differences were found, populations at the time of necropsy in almost all passages were larger than those on days 3 or 4 and sometimes larger than those on days 9 or 10. For strain 11168, CYTH4 population sizes on day 3 or 4 were significantly different from those both on day 9 or 10 and at the time of necropsy (Pcorrected = 0.01 and 0.02, respectively); population sizes on day 9 or 10 were not significantly different from those at the time of necropsy. Furthermore, significant differences in fecal population sizes between passages were found for strains 11168, D2600, and NW. For strain 11168, the comparison between passages was significant for the comparison of passage 1 to both passages 2 and 3 (Pcorrected = 6.8 × 10-7 and 6.0 × 10-8, respectively) and for the comparison of passages 2 and 3 (Pcorrected = 1.2 × 10-3). For strains D2600 and NW, only the comparison between passages 1 and 3 was significant (Pcorrected = 7.4 × 10-4 and 0.017, respectively). The fraction of mice harboring C. jejuni in the jejunum also increased over the serial passage experiments for strains 11168, D0835, and D2600 (Additional file 1, Table S1).